• 한국수정란이식학회지
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• 제9권1호
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• pp.85-93
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• 1994

This study was carried out to investigate the effects of gonadotropins added during maturation of porcine oocytes on the in vitro maturation(IVM), in vitro fertilization(IVF) and developmental potential of embryos. The follicular oocytes were cultured in TCM-199 medium containing different combination of gonadotropins(5$\mu$g /ml FSR or 1OIU /ml PMSG and 1O$\mu$g /ml LH or 1OIU /ml hCG), 10% FCS and 10% PFF for 36~48h in a incubator with 5% $CO_2$ in Air at 39$^{\circ}C$ and then matured oocytes were again cultured to 120h after IVF for 6~7h with heparin(100$\mu$g /m')-treated sperm. When the oocytes were matured for 42brs in the medium containing FSH+LH, FSH+hCG, PMSG+LH or PMSG+hCG, the JVF rate of each treatment was 50.0%, 52.9%, 66.7% and 70.0%, respectively. The highest CEI (cumulus cell expansion index) was obtained from PMSG+hCG-added medium and the highest polyspermic penetration resulted from FSH+LH-added medium. The cleavage of IVF oocytes derived from hormone added IVM was significantly(P<0.05) promoted by PMSG+hCG and the cleavage rate after 36-h, 42-h and 48-h maturation aws 53.0%, 56.7% and 45.6%, respectively. The highest developmental potential resulted from the oocytes derived from PMSG+LH -added IVM.

• Reproductive and Developmental Biology
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• 제28권1호
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• pp.13-20
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS, and primary fibroblast cultures were established in TCM-199 with 10% FBS. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3% hyaluronidase. The matured oocytes were dipped in D-PBS plus 10% FBS＋7.5 $\mu\textrm{g}$/ml cytochalasin B and 0.05 M sucrose. The reconstructed oocytes were electrically fused with donor cells in 0.3 M mannitol fusion medium. After the electofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. On the other hand, the NT embryos with porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6∼8 day at $39^{\circ}C, 5% CO_2$ in air. In caprine-bovine NT embryos, the cleavage(2-cell) rate was 36.8% in confluence and 43.8% in serum starvation. The developmental rate of morula- and blastocyst-stage embryos was 0.0% in confluence and 18.8% in serum starvation. In caprine-porcine NT embryos, the cleavage(2-cell) rate was 76.7% in confluence and 66.7% in serum starvation. The developmental rate of morula and blastocyst stage embryos was 3.3% in confluence and 3.0% in serum starvation, and no significant difference was observed in synchronization treatment between donor cells. In caprine-bovine NT embryos, the cleavage(2-cell) rate of cultured donor cells was 30.8% and 17.6% in 5∼9 and 10∼14 passage(P＜0.05). The developmental rate of morula and blastocyst stage embryos were significantly higher(P＜0.05) in 5∼9 passage(23.1%) than in 10∼14 passage(0.0%) of cultured donor cells. In caprine-porcine NT embryos, the cleavage rate was significantly higher(P＜0.05) in 5∼9 passage(86.7%) than in 10∼14 passage(50.0%) of cultured donor cells. The developmental rate of morula and blastocyst stage embryos were 3.3 and 0.0% in 5∼9 and 10∼14와 passage of cultured donor cells. In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer, 33.9% in in vitro fertilization and 28.1% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than in vitro fertiltzation(26.9%) and parthenotes(37.4%).

• Reproductive and Developmental Biology
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• 제32권4호
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• pp.249-253
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for studying the interactions between an oocyte and somatic nucleus. The object of this study was to investigate the developmental competence of in vitro-matured porcine oocytes after transfer of the somatic cell nuclei of 2 different species (goat and rabbit). Porcine cumulus oocytes were obtained from the follicles of ovaries and matured in TCM-199. The reconstructed embryos were electrically fused with 2 DC pulses of 1.1kV/cm for $30{\mu}s$ 0.3M mannitol medium. The activated cloned embryos were cultured in porcine zygote medium-3 (PZM-3), mSOF or RDH medium for 7 days. The blastocyst formation rate of the embryos reconstructed from goat or rabbit fetal fibroblasts was significantly lower than that of the embryos reconstructed from porcine fetal fibroblast cells. However, a significantly higher number of embryos reconstructed from goat or rabbit fetal fibroblasts cultured in mSOF or RDH, respectively, developed to the morular stage than those cultured in PZM-3. These results suggest that goat and bovine fetal fibroblasts were less efficacious than porcine-porcine cloned embryos and that culture condition could be an important factor in iSCNT. The lower developmental potential of goat-porcine and porcine-bovine cloned embryos may be due to incompatibility between the porcine oocyte cytoplasm and goat and bovine somatic nuclei.

• Asian-Australasian Journal of Animal Sciences
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• 제31권6호
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• pp.827-834
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• 2018

Objective: The aim of this study was to compare the effects of $36.5^{\circ}C$ and $38.5^{\circ}C$ incubation temperatures on the maturation of bovine oocytes and developmental competence of embryos. Methods: In experiment 1, oocytes were maturated in bicarbonate-buffered TCM-199 for 22 hours in a humidified atmosphere of 5% $CO_2$ in the air at either $36.5^{\circ}C$ or $38.5^{\circ}C$ and nuclear maturation status were determined. In experiment 2, in vitro fertilized oocytes were allocated randomly into synthetic oviductal fluid medium with or without a mixture of 1 mM L-glutathione reduced and 1,500 IU superoxide dismutase and cultured in a humidified atmosphere of 5% $CO_2$, 5% $O_2$, and 90% $N_2$ in the air at $38.5^{\circ}C$ for 8 days. Results: There were no significant differences between incubation temperatures in terms of oocyte maturation parameters such as cumulus expansion, first polar body extrusion and nuclear maturation. Incubation temperatures during in vitro maturation had no effects on developmental competence of embryos, but supplementation of antioxidants increased (p<0.05) developmental competence of the embryos. Blastocysts from oocytes matured at $38.5^{\circ}C$ had comparatively higher inner cell mass, but low overall and trophectoderm cell numbers (p<0.05). Conclusion: The results of present study showed that maturation of bovine oocytes at $36.5^{\circ}C$ may provide a suitable thermal environment for nuclear maturation and subsequent embryo development.

• 한국발생생물학회지:발생과생식
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• 제21권2호
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• pp.205-213
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• 2017

The aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and $100{\mu}M$) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with $50{\mu}M$ ALA were fertilized and cultured in IVC medium with ALA (25, 50 and $100{\mu}M$) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with $25{\mu}M$ ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by $50{\mu}M$ ALA treatment groups compared with control groups (p<0.05). Treatment of $25{\mu}M$ ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by $25{\mu}M$ ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.

• Reproductive and Developmental Biology
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• 제37권2호
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• pp.57-64
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• 2013

Developmental potential of cloned embryos is related closely to epigenetic modification of somatic cell genome. The present study was to investigate the effects of applying histone deacetylation inhibitor, trichostatin A (TSA) to activated porcine embryos on subsequent development of porcine parthenogenetic and nuclear transfer embryos. Electrically activated oocytes were treated with 5 nM TSA for different exposure times (0, 1, 2 and 4 hr) and then the activated embryos were cultured for 7 days. The reconstructed embryos were treated with different concentrations of 0, 5, 10 and 25 nM TSA for 1 hr. Also 5 nM TSA was tested with different exposure times of 0, 0.5, 1, 2 and 4 hr. And fetal fibroblast cells were treated with 50 nM TSA for 1, 2 or 4 hr and with 5 nM TSA for 1 hr. Cumulus-free oocytes were enucleated and reconstructed by TSA-treated donor cells and electrically fused and cultured for 6 days. In parthenogenetic activation experiments, 5 nM TSA treatment for 1 hr significantly improved the percentage of blastocyst developmental rates than the other groups. Total cell number of blastocysts in 1 hr group was significantly higher than other groups or control. Similarly, blastocyst developmental rates of porcine NT embryos following 5 nM TSA treatment for 1 hr were highest. And the reconstructed embryos from donor cells treated by 50 nM TSA for 1 hr improved the percentage of blastocyst developmental rates than the control group. In conclusion, TSA treatment could improve the subsequent blastocyst development of porcine parthenogenetic and nuclear transfer embryos.

• 한국수정란이식학회지
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• 제9권1호
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• pp.117-125
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• 1994

In vitro development of parthenogenetic embryo was examined after ethanol treatment of follicular oocytes matured in vitro for 42, 48, 54 and 60h in the pig. The follicular oocytes were matured in TCM 199 containing 15% FCS and gonadotrophins in an atmosphere of 39 $^{\circ}C$ 5% $CO_2$. The cumulus-free oocytes were activated by 10% ethanol treatment in M2+4mg /ml BSA for 10 min. The ethanol-activated oocytes were washed and further cultured in TCM199+20%FCS containing granulosa cell monolayer. Maturation rates at 42, 48, 54 and 60h of IVM were 75.0, 86.5, 81.6 and 87.9%, respectively. Thus the oocytes maturated in vitro for longer periods did not improve nuclear maturation much. Pronuclear formation rates at 18h post-activation in ethanol-activated oocytes were 21.9, 25.0, 47.4 and 32.6%. The cytoplasmic maturation leading to pronuclear formation upon activation increased when the I VM period was extended from 42 to 54h. When the activated oocytes were cultured for 96~120h to analyse early development of the activated oocytes, the rates of embryonic development upto $\leq$ 5~8 cell were 5.3, 5.8, 12.0 and 11.7% among the cultured embryos. The result indicate that earlier development of activated porcine occyte is dependent on the duration of oocyte maturation, and that better development could be obtained from the oocyte matured for 54h.

• 한국가축번식학회지
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• 제17권2호
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• pp.103-111
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• 1993

These experiments were undertaken to establish the optimal culture systems for in vitro maturation, fertilization and subsequently embryonic development of porcine immature follicular oocytes isolated from the ovary of slaughtered pigs. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles (3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated (39$^{\circ}C$, 5% CO2 in air) for 42hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were prepared forfertilizaing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${mu}ell$ of capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the culture media. The fertilization rates of in vitro matured follicular oocytes cultured in B. O., TCM-HEPES, m-KRB, and TALP-II media were 61.3%, 83.0%, 88.9% and 89.2%, respectively. In addition, the polyspermy rates were 60.7%, 66.5%, 53.8%, and 43.9%, respectively. These data indicated that the highest of fertilization and the lowest of polyspermy rate was shown in TALP-II medium. Spermatozoa capacitated by caffeine, heparin, and percoll density gradient treatment in the 4 different media, the fertilization rates were 33.0~57.2%, 39.9~90.2%, and 52.6~92.8%, respectively, showing the lowest rate in caffeine treatment. The development rate of follicular oocytes, fertilized with the spermatozoa capacitated by caffeine, heparin, and percoll gradient in the TALP-II medium, upto 2 to 4-cell stages were 32.6%, 74.5% and 70.9%, respectively. Finally, fertilization rates of follicular oocytes cultured with follicular fluid containing medium from 10 to 100% were 61.2~94.1% and the rates (90~94%) with 10~20% follicular fluids were significantly higher than those (85.3%) of cultured in the media without follicular fluid. In addition, the rates of pronucleus formation were also higher in follicular fluid treated group (73.1~83.0%) than those (64.7%) of oocytes cultured without follicular fluid. The highest fertilization and pronucleus formation rates was found in oocytes cultured with 10% follicular fluid. These results suggest that the addition of heparin or percoll density gradient method is better capacitation method. Furthermore, the addition of porcine follicular fluid to the fertilization medium may improve the fertilization rates and formation of pronucleus.

• Asian-Australasian Journal of Animal Sciences
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• 제24권3호
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• pp.336-343
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• 2011

The neurotrophins, required for the survival and differentiation of the nervous system, are known to be important for the development of the reproductive tissues. However, the signals initiating the growth of follicles, gamete development, and transport and the development of zygote in the reproductive system of cows remain ambiguous. The purpose of the present study was to identify the transcripts and proteins of Neurotrophin 4 (NT4) and its receptor tyrosine kinase B (TrkB) in bovine reproductive tissues. The transcripts and immunoreactivity of NT4 and TrkB proteins were detected by reverse transcription polymerase chain reaction and western blot analysis. Using immunohistochemistry, the specific immunoreactivity of NT4 and TrkB were detected in the oocytes of primordial follicles and in the growing primary follicles. The NT4 and TrkB immunoreactivity was predominantly observed in granulosa cells, cumulus granulosa cells, cumulus oocyte complexes, theca cells of mature follicles, as well as in the oviduct epithelial cells, uterine gland cell, and epithelium cells of the uterus during the follicular and luteal phases in cows. Expressions of NT4 and TrkB mRNAs were not significantly different among the ovary, oviduct, and uterus of the follicular phase. For the luteal phase, the expression of NT4 mRNA in the ovary was significantly higher than that in the oviduct and uterus, and the expression of TrkB mRNA in the oviduct was significantly higher than that in the ovary and uterus, as determined by fluorescence quantitative reverse transcription polymerase chain reaction. The expression of NT4 mRNA was significantly higher than that of TrkB mRNA in the ovary and uterus, whereas NT4 mRNA expression was lower than that of TrkB mRNA in the oviduct during the luteal phase. The present study hypothesizes that NT4 participates in the regulation of both gonads and extra-gonadal reproductive tissues in cows.

• 한국수정란이식학회지
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• 제29권3호
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• pp.201-206
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• 2014

The objectives of the study were to determine an effective culture dish, culture duration and protein supplementation in medium for in vitro maturation (IVM) of oocytes of native zebu cows in Bangladesh. The ovaries of cows were collected from local slaughterhouse followed by aspiration of follicular fluid. The cumulus-oocyte-complexes (COCs) with more than 3 compact cumulus cell layers were cultured in tissue culture medium (TCM) 199 for maturation. The maturation of oocytes was determined by observing polar body under microscope. To determine an effective culture dish, 130 COCs derived from 48 ovaries in a well of 4-well dish and 102 COCs derived from 36 ovaries in drops covered with mineral oil within 35 mm petri dish were cultured for 24 hours. The rate of maturation of oocytes did not vary between 4-well dish ($51.3{\pm}15.0%$) and drops in petri dish ($52.4{\pm}11.6%$). To determine the effective culture duration, 185 COCs derived from 62 ovaries were cultured in drops for 18, 21, 24 and 27 hours. The rate of maturation of occytes ranged from $51.9{\pm}9.4%$ (18 hours) to $59.0{\pm}17.1%$ (27 hours) and the difference in maturation rate among different culture durations was not significant (P>0.05). To determine an effective protein supplementation, 63 oocytes from 19 ovaries were cultured separately in TCM 199 supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). The rate of maturation was significantly (P<0.01) higher in medium supplemented with FBS ($55.63{\pm}16.19%$) than that of BSA ($14.82{\pm}9.36%$). In conclusion, COCs of native zebu cows can be cultured for IVM either in 4-well culture dish or droplets in petri dish for 18 to 27 hours in medium supplemented with FBS.